Maternal folic acid, the synthetic form of folate, is one of the first modifiable factors identified to date with the potential to reduce occurrence of autism spectrum disorders (ASD) by 40% if taken near conception. Folic acid appears to protect against ASD especially in mothers and children who are genetically susceptible to inefficient folate-dependent one-carbon and methylation metabolism, but this finding needs replication. In addition to being essential for neurodevelopment, folate is a primary methyl-donor for methylation reactions, including DNA methylation. The time near conception is an especially critical period for adequate methyl supply during cycles of active demethylation and re-methylation of the genome during embryogenesis. Our preliminary data show that folic acid supplementation is associated with even greater reductions in ASD risk, by 75%, in younger siblings of children with ASD. In addition, we found DNA methylation differences associated with no maternal use of folic acid supplements in birth tissues (placenta and cord blood) for genes with nuclear regulatory and brain development functions that could have implications for ASD. The goal of the proposed work is to leverage data and samples from mother-child pairs in two large prospective pregnancy cohorts of high-risk infant siblings to examine specific pathways for prevention of ASD through maternal dietary and supplemental folate intake. This work will build on previous studies by examining exposures collected prospectively, with more accuracy in terms of timing and dose. We will be first to examine whether other methyl-donor B-vitamins have associated effects. We will include information on dietary and supplemental folate and B-vitamin intake from validated instruments, and measurements from maternal first trimester serum, cord blood, and fetal placental tissue. We will examine folic acid interactions with genetic susceptibility factors. Finally, we will increase understanding of the underlying mechanisms by identifying DNA methylation changes associated with folate status, and investigate whether these changes overlap with methylation patterns observed in autistic brains, using innovative methods, multiple platforms, and replication across tissue type and study population, to address challenges associated with DNA methylation measurement. Through completion of the proposed project, we will gain a better understanding of how strongly folic acid is associated with reduced risk of ASD in high-risk families, when intake is most associated and at what levels, how this association differs based on genetic susceptibility, and whether maternal folic acid intake alters DNA methylation profiles in ways that could decrease ASD risk. These findings will have great clinical and public health implications, informing autism prevention trial and ultimately changes in recommendations and policy.